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SAM 912.03

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United States Department of Agriculture

Center for Veterinary Biologics

Testing Protocol

SAM 912

Supplemental Assay Method for the Determination of Total Bacterial Count 

in Antibody Products

Date: February 21, 2023

Number: SAM 912.03

Supersedes: SAM 912.02, January 12, 2017

Standard Requirement: 9 CFR 113.450(h)(2)(iv)

Contact: Email: Methodsrequest.notification@usda.gov

 Phone: Center for Veterinary Biologics, 515-337-6100

United States Department of Agriculture

Animal and Plant Health Inspection Service

P. O. Box 844

Ames, IA 50010

Mention of trademark or proprietary product does not constitute a guarantee or warranty of the product 

by USDA and does not imply its approval to the exclusion of other products that may be suitable.
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Table of Contents

1. Introduction

2. Materials

2.1 Equipment/instrumentation

2.2 Reagents/supplies

3. Preparation for the Test

3.1 Personnel qualifications/training

3.2 Preparation of equipment/instrumentation

3.3 Preparation of reagents/control procedures

3.4 Preparation of the sample

4. Performance of the Test

5. Interpretation of the Test Results

6. Record and Report of Test Results

7. References

8. Summary of Revisions

Appendix
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1. Introduction

This Supplemental Assay Method (SAM) describes the test procedure used to determine 

the total bacterial count in dried and liquid antibody products for oral administration, per

title 9, Code of Federal Regulations (9 CFR), part 113.450(h)(2)(iv). This test procedure

uses Tryptone glucose extract agar (TGEA) to determine the colony-forming units (CFU)

of contaminating bacteria.

2. Materials

2.1 Equipment/instrumentation

Equivalent equipment or instrumentation may be substituted for any brand name listed 

below.

2.1.1 Laminar-flow Class II biosafety cabinet (BSC)

2.1.2 Lab Armor® bead bath (set to 50°- 55°C)

2.1.3 Vortex mixer

2.1.4 30°- 35°C incubator

2.1.5 Analytical balance

2.1.6 Colony counter

2.2 Reagents/supplies

Equivalent reagents or supplies may be substituted for any brand name listed below.

2.2.1 Bacillus subtilis (American Type Culture Collection (ATCC) #6633) or 

equivalent positive control culture

2.2.2 Tryptone glucose extract agar (TGEA) (Appendix), National Centers for 

Animal Health (NCAH) Media #50072 or as stated in the Outline of Production

(OP) from the biologics manufacturer.

2.2.3 Sterile water 

2.2.4 Sterile pipettes, individually packaged

2.2.5 Sterile petri dishes, 100 x 15-mm
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2.2.6 Sterile culture tubes 

2.2.7 Lab coat or sterile sleeves and gloves

2.2.8 Spatulas

2.2.9 Weigh boats

2.2.10 Sterile wide mouth snap cap specimen containers

2.2.11 70% ethanol

2.2.12 4 x 4-inch sterile gauze pads

2.2.13 Pipetting aid

2.2.14 Micropipettors, 100-μL to 1.0-mL

2.2.15 Pipette tips, 100-μL to 1.0-mL

2.2.16 Sterile syringes with needles

3. Preparation for the Test

3.1 Personnel qualifications/training

Personnel must have experience or training in this protocol. This includes knowledge 

of aseptic biological laboratory techniques and preparation, proper handling and 

disposal of biological agents, reagents, tissue culture samples, and chemicals.

Personnel must also have knowledge of safe operating procedures and policies, as

well as training in the operation of the necessary laboratory equipment listed in 

Section 2.1.

3.2 Preparation of equipment/instrumentation

3.2.1 Operate all equipment and instrumentation according to the 

manufacturer’s instructions and maintain according to standard operating 

procedures (SOPs).

3.2.2 Turn on the BSC at the beginning of the work week and leave on all week.

3.2.3 Turn on the bead bath at least one day before use to allow the temperature 

to equilibrate. 
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3.2.4 Monitor temperature of incubators, freezers, coolers, and bead baths 

according to SOPs.

3.3 Preparation of reagents/control procedures

3.3.1 Warm the samples and reference culture to room temperature before 

rehydrating, if needed, to the appropriate volume.

3.3.2 Bacillus subtilis reference stock culture is prepared according to the

manufacturer’s instructions and titrated to determine the CFU concentration. For 

each test session, inoculate two petri dishes with a volume containing 

approximately 100 CFU to serve as a positive control.

3.3.3 Negative/Technique Control: Inoculate one petri dish with 1 mL of the

water that was used in the testing session. If no diluent is used in the testing

session, use sterile water packaged in serum vials as the inoculum. Pour 15-20

mL of molten TGEA into each plate, swirl gently to mix and allow to solidify. 

Once the agar has hardened, invert the petri dish. Incubate this control plate 

with the serial test plates.

3.3.4 Preparation of the TGEA medium: On the day of the test, melt the TGEA 

in an autoclave for 30 minutes at 100°C. Place the bottles of melted media in the

bead bath. Do not begin testing until the agar has cooled to at least 55°C. The 

temperature of the bead bath should be approximately 50°- 55°C when the agar is 

ready. 

 3.4 Preparation of the sample

3.4.1 At least ten containers of final product are required for total bacterial 

count purity testing.

3.4.2 Check the firm’s OP, Section V, to obtain the maximum CFUs allowed in

the product before determining the dilution scheme. 

3.4.3 Samples are liquid or dried antibody products. When needed, sterile 

purified water in volumes specified on the product label or the diluent specified 

in the firm’s OP is used for rehydrating samples.

3.4.4 Weigh samples of product on an analytical balance, if needed. Rehydrate 

samples of the product according to label or instructions from the firm's OP. 

Dissolve samples by stirring on a stir plate or shake with a vortex mixer. If 

sample does not readily go into solution after manipulating, allow the diluted 

sample to sit in a 30°- 35°C incubator for a minimum of 30 minutes. 

 
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 3.4.5 Label 10 plates for each serial with the sample number or name and the 

 container number.

 4. Performance of the Test

4.1 Dress in a clean lab coat or sterile sleeves and gloves to perform purity testing.

4.2 Wipe down the interior surfaces of the BSC used for testing with 70% ethanol 

immediately prior to use and between testing each serial.

4.3 Place the necessary testing materials (micropipettors, pipet tips, 4 x 4-inch gauze 

squares, petri dishes, etc.) and the diluted sample into the BSC.

4.4 Mix the sample thoroughly. Dispense 1 mL of inoculum from one sample 

container into a petri dish. 

4.5 Repeat Section 4.4 for the other nine sample containers for this serial.

4.6 Dispense 15-20 mL of TGEA medium into each of the 10 dishes for this serial.

Swirl gently to mix and then allow the agar to cool in the BSC. Once the agar has 

hardened, invert the petri dishes and place the dishes in a 30°- 35°C incubator for up 

to 48 hours.

4.7 Repeat Sections 4.3 through 4.6 for the other serials of biologic to be tested.

4.8 Once all the test serials have been put on test, prepare the negative/technique

controls for the testing session (see Section 3.3.3). Prepare negative controls using sterile 

water if no diluent was used in the testing session. Dispense 1 mL of water into a petri 

dish and pour 15-20 mL of TGEA into the plate. Swirl gently to mix and allow the agar 

to cool in the BSC. Once the agar has hardened, invert the petri dish.

4.9 Once the purity portion of the test has been completed, prepare the positive 

control organism in an area that is separate and apart from the clean area where the purity 

test was conducted (see Section 3.3.2). 

4.9.1 Inoculate two petri dishes with approximately 100 CFUs of B. subtilis. 

4.9.2 Dispense 15-20 mL of TGEA medium into each plate. Swirl gently to mix 

and then allow the agar to cool in the BSC. Once the agar has hardened, invert the 

petri dishes.

4.10 All plates are incubated at 30°- 35°C for up to 48 hours.
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5. Interpretation of the Test Results

5.1 Criteria for a valid test:

5.1.1 There must be no growth in the negative/technique control and media

control plates.

5.1.2 The positive control plates at the Center for Veterinary Biologics must 

contain an average count of 81-112 CFU.

Note: A range of approximately 100 CFU should be determined at each biologics 

manufacturer facility for each new positive control lot.

5.1.3 If these criteria are not met, the test is considered invalid or a no test (NT). 

Products may be reported and released with a NT result if there is no reason to 

suspect an unsatisfactory (UNSAT) sterility result for that product.

5.2 After incubation, all plates are examined. Plates that contain 30 to 300 bacterial 

colonies are counted with a colony counter. Determine the average count for the 

10 plates counted. 

5.3 Compare the average counts to the maximum CFU allowed in Section V of the 

firm's OP. If the average count is less than or equal to the maximum CFU allowed, the 

product is satisfactory (SAT) for the total bacterial count. If the average count is larger 

than the maximum CFU, the test is considered inconclusive (INC) so one retest (RT) to 

rule out faulty technique may be conducted. If a RT is not conducted within 21 days, the 

product is UNSAT by the first test.

5.4 If growth is observed on the negative or media control plate(s), or no growth is 

observed on the positive control plates, the test is a NT and may be repeated.

5.5 If a RT is ordered, double the number of samples used in the first test are 

required. If the average total count of the 20 plates from the RT is larger than the 

maximum CFU allowed, the serial is UNSAT.

6. Record and Report of Test Results

Record and report results of the test(s) according to SOPs.

7. References

Title 9, Code of Federal Regulations, part 113.450(h)(2)(iv), U.S. Government Printing 

Office, Washington, DC.
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Standard methods for the examination of dairy products. 1992, 16th Edition, Robert T. 

Marshall, editor. American Public Health Association, Washington, D.C. 

8. Summary of Revisions 

Version .03 

• The coversheet and the formatting have been updated. 

Version .02 

• Section 5.1.2 was rewritten for clarification.
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Appendix

Tryptone Glucose Extract Agar (TGEA) – National Centers for Animal Health (NCAH)

 Media #50072

Tryptose Glucose Extract Agar 24.0 g

QH20 1000.0 mL

Autoclave 20 minutes at 121°C. Store at 2°- 5°C for up to 3 months.